Solubleproteinhasneverbeenthisfastandeasy!
- Enzyme-freedirectionalPCRcloninginseconds!
- Savedaysofeffortwithready-to-usevectorsandcompetentcells-NOligationstep.
- Tightly-controlledexpressionofN-terminal6xHis-taggedproteinswithcleavableSUMOsolubilitytag.
- SystemsavailablewithexpressionunderthecontrolofT7ortunableRhamnosepromoters.
CompleteCloningandProteinExpressionSystem
TheExpressoSUMOCloningandProteinExpressionSystemsaredesignedforfast,easy,andefficientdirectionalcloningandsolubleexpressionofPCR-amplifiedgenes.Forproteinsthatforminclusionbodiesoraredifficulttoexpressinsolubleform,thepETite®N-HisSUMOandpRham™N-HisSUMOVectorsallowexpressionoftargetproteinswithanamino-terminal6xHis-SUMOproteintag.SUMO(smallubiquitin-likemodifier)isarelativelysmall(100-residue)polypeptidethathasbeenshowntoenhancethesolubleexpressionofmanyproteinsthatareotherwisedifficulttoproduceinE.coli.
TheExpressoSUMOCloningandProteinExpressionSystemsarebasedontheoriginalExpressoT7andExpressoRhamnoseCloningandProteinExpressionSystems,whichuseExpressioneeringTechnology™toprovideeffortlesshigh-efficiencycloningandtightlycontrolledproteinexpression.TheT7Systemiscompletewithpre-processedpETiteN-HisSUMOcloningvector,andtwocompetentcelllines,suppliedinsingletransformationvials.HighefficiencyHI-Control™10GChemicallyCompetentCellsenablestablecloningandHI-ControlBL21(DE3)CompetentCellsprovidetightlycontrolledproteinexpression,thushelpingyouavoidproteinexpressionproblemsseenwithleakyT7promoter-basedexpressionplasmidsystems.TheRhamnoseSystemiscompletewithpre-processedpRhamN-HisSUMOcloningvectorandhigh-efficiencyE.cloni10GCompetentCells,whichareusedforcloningandexpression,enablinghigherproteinexpressionthroughput.TheN-terminal6xHisSUMOtaggedrecombinantproteinscanberapidlypurifiedusingstandardNickelchromatography.SUMOExpressProteaseisincludedtoprovideefficientcleavageoftheSUMOtag,preciselyatthejunctionbetweentheSUMOtagandthetargetprotein.
Five-SecondDirectionalCloningofPCR-AmplifiedGenes
TheExpressoSUMOCloningandProteinExpressionSystemsuseExpressioneeringTechnology,theenzyme-freerecombinationalcloningstrategyoftheExpressosystemtoseamlesslyintegratethegenewiththeexpressionplasmid.ThetargetgeneisamplifiedbyPCRusingprimersthatadd18base-pairsofvector-complementarysequencetobothendsofthegene.Unlikeotherrestrictionenzymebasedmethodsorligase-freecloningmethods,nofurthercleanuporenzymatictreatmentofthePCRproductisnecessary.Simplymix1µloftheunpurifiedPCRreactionwiththesuppliedpre-processedpETiteorpRhamN-HisSUMOexpressionplasmids,andtransformimmediatelyintotheChemicallyCompetentCellsprovided(Figure1).
SUMOVectorsincludes:
StrongT7promoterforhigh-level,orRhamnosepromoterfortunable,recombinantproteinexpression.
IncreasedsolubilityandfastproteinpurificationfromN-terminal6xHisSUMOfusiontag
Smallsize(2.5kb)foreasierdownstreammanipulation.
PatentedCloneSmart®technologyincreasescloningefficiency.
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Figure2.SUMOexpressionvector:Smallsize(2.5kbvs.5.4kbforpET)foreasiermanipulation,includingtargetedmutagenesis.Expressionplasmidispre-processedforinstantenzyme-freecloningofPCRproducts. |
RescueinclusionbodiesandinsolubleproteinwithSUMOproteintag:
WehaveusedtheExpressoT7andExpressoT7SUMOCloningandExpressionSystemsforexpressionandpurificationofavarietyofproteins.Someresultsofanongoinglarge-scaleexpressionstudytoidentifyhydrolaseenzymesfromFibrobactersuccinogenesarepresentedinFigure3.Initially,48geneswereselectedforexpressiontrialsandclonedintothepETiteT7C-HisVector.Approximatelyhalfofthesecloneshaveproducedsoluble,activehydrolaseprotein,whileinotherinstancestargetproteinswereexpressedinaninsolubleform.FiveofthegenesproducinginsolubleproteinswerereamplifiedandclonedintothepETiteSUMOvector.WhentheresultingcloneswereexpressedinHI-ControlBL21(DE3)cells,recoveryofactiveproteininthesolublefractionwassignificantlyimprovedinfourofthefivecases(Table1).Althoughtagremovalwasnotnecessaryforhydrolaseactivity,thetagcouldberemovedefficientlybySUMOExpressProtease.
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Figure3.Large-scalecloningandexpressioncasestudy:(A)PCRproductsfrom48putativehydrolasegenesrangingfrom~1to>3kb.(B)Uninduced(-)andIPTG-induced(+)samplesofHI-ControlBL21(DE3)cellswith6differentgenesclonedintothepETiteC-HisVector.(C)EnhancedsolubilityofSUMO-tagged2201and2442geneproducts.Totalcellextractandsolublefractionsareshown.(D)Removalof6xHis-SUMOtagfrompurifiedSUMO-2201fusionproteinbySUMOprotease.–prot:uncleavedSUMO-2201fusionproteinafterIMACpurification;+prot:SUMOprotease-treatedfusionprotein;C:isolated2201proteinafterremovalof6xHis-SUMOfragmentandSUMOproteasebysubtractiveIMAC. |
Fibrobactersuccinogenes | Solubleproteinyield | |
w/oSUMOtag | w/SUMOtag | |
1425 | 0mg/liter | 0mg/liter |
1765 | 0mg/liter | 10mg/liter |
1793 | 0mg/liter | 17mg/liter |
1994 | 0mg/liter | 17mg/liter |
2201 | 0mg/liter | 20mg/liter |
Table1. ImprovementofsolubleproteinyieldwithSUMOtag.Yieldofsolubleproteinwasimprovedsignificantlyfor4of5FibrobactersuccinogenesgeneswhenclonedintopETiteN-HisSUMOandexpressedinHI-ControlBL21(DE3)Cells.Cultureswereinducedwith1mMIPTGandgrownovernightat22°C.Yieldswerecalculatedfromtheamountofpureproteinobtainedfrom100mlofcellcultureafterpurificationoveraNi-NTAcolumn.
CleavageofSUMOproteintag
AfterIMACpurificationoftheN-His-SUMOtaggedprotein,thetagportioncanberemovedpreciselybytheincludedSUMOExpressProtease.TheSUMOExpressProteaserecognizesthetertiarystructureofSUMOratherthanashortrecognitionsequenceandcleavespreciselyatthejunctionbetweentheSUMOtagandthetargetprotein,withnooff-targetcleavage.BoththeSUMOExpressProtease,whichis6xHistagged,andthecleavedN-His-SUMOtagcanthenbeseparatedfromthereleasedtargetproteinbysubtractiveIMAC.
Note:TheSUMOproteintagincludedinthesekitsisaspeciallyengineeredversionofSUMOthatcanbecleavedonlyusingLucigen'sSUMOExpressProtease.SUMOExpressProteasedoesnotcleavethewildtypeSUMOsubstrateatanyusefullevel.Therefore,itisnotrecommendedtouseSUMOExpressoProteasetocleavewildtypeSUMO.
ImportantProductUseInformation
ThisproductisthesubjectofU.S.Patent#6,709,861.AdditionalpatentapplicationsownedbyLucigenCorporationarepending.
The6xHistagislicensedfromHoffmann-LaRoche,Inc.,Nutley,NJand/orHoffmann-LaRocheLtd.,Basel,Switzerlandandisprovidedonlyfortheuseinresearch. InformationaboutlicensesforcommercialuseisavailablefromQiagenGmbH,QIAGENStrasse1,D-40724Hilden,Germany.Purificationof6xHistaggedproteinswithNi-NTAmanufacturedbyQIAGENishighlyrecommendedforbestperformancesandtoavoidpoorpurificationresults.
SUMOExpressProteaseismanufacturedandsuppliedbyLifeSensors,Inc.
ORDERINFORMATION
TheExpressoT7SUMOCloningandExpressionSystemcontainspre-processedpETite®N-HisSUMOVectorDNA,HI-Control™10GChemicallyCompetentCellsforcloning,andHIControlBL21(DE3)ChemicallyCompetentCellsforproteinexpression.AlsoincludedareSUMOPositiveControlCInsertDNA,transformationpositivecontrolpUCDNA,SUMOExpressProtease,SUMOCleavageControlProtein,andforwardandreversePCRprimerstoconfirmclones.
TheExpressoRhamnoseSUMOCloningandExpressionSystemcontainspre-processedpRhamN-HisSUMOVectorDNA,single-transformationE.cloni10GChemicallyCompetentCells(SOLOs),andtheauto-inductionreagents20%Rhamnosesolutionand15%Glucosesolution.AlsoincludedareSUMOPositiveControlCInsertDNA,transformationpositivecontrolpUCDNA,SUMOExpressProtease,SUMOCleavageControlProtein,andforwardandreversePCRprimerstoconfirmclones.
